Carcinoembryonic antigen, CEA, was first described in 1965 as cancer-specific fetal antigen in adenocarcinoma of the human digestive tract. In 1986, the CEACAM5 gene (carcinoembryonic antigen cell adhesion molecule 5) that encodes human CEA was localized to chromosome 19, and in 1987 it was cloned.
CEA is a 180 kD glycoprotein that is anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) lipid moiety. CEA is heavily glycosylated, approximately 60% by weight (Egan, M. L. et al. (1976) Cancer Research 36:3482-3485). CEA has only been found in humans. CEA has many family members, including many cross-reacting antigens, some of which have been found in other species. CEA protein is found at high levels in the fetal colon and lower levels in the normal adult colonic epithelium. Levels of CEA can become elevated when inflammation or tumors arise in any endodermal tissue, including in the gastrointestinal tract, respiratory tract, pancreas, and breast. CEA is present at abnormally high levels in several benign disorders and in malignant tumors, including those of the stomach, small intestine, colon, rectum, pancreas, liver, breast, ovary, cervix, and lung. Non-malignant disorders showing high levels of CEA include: diverticulitis, pancreatitis, inflammatory bowel disease, cirrhosis, hepatitis, bronchitis, and renal failure. CEA is also elevated in individuals who smoke.
The function of CEA in normal colon epithelial cells is still being investigated. It is thought that CEA, which is localized on the cell surface, acts as a homotypic adhesion molecule, resulting in aggregation of CEA-expressing cells. The pattern of localization is different in colon tumor cells compared to normal colon cells. In the normal colonocytes, CEA is localized only at the luminal surface of cells, whereas in tumor cells, it is found in a disoriented pattern throughout the cell membrane. The altered pattern in tumor cells may disrupt the intercellular adhesion resulting in the disorganized growth and movement of malignant cells. CEA and its closely related family members have also been shown to participate in signal transduction. In normal human colon, CEA is released from cells via pinching off of CEA-coated vesicles. More than 90% of total CEA in normal feces is membrane bound. It has also been theorized that CEA might play a role in innate immunity by binding and trapping microorganisms in the digestive tract and other tissues (Frangsmyr, L. et al. (1999) Tumor Biology 20:277-292 and Hammarstrom, S. and Baranov, V. (2001) Trends in Microbiology 9(3):119-125). This binding would prevent the microorganisms from reaching and invading epithelial cells of the microvilli. The constant and rapid release of CEA-containing vesicles could provide a method for constant trapping and rapid release of bacteria.
The CEA gene (Genbank Accession Number M17303) encodes an mRNA of 3100 nucleotides and translates into a 702 amino acid protein that is cleaved of a 34-amino-acid signal sequence to form a 668 amino acid protein of 70 kD (Oikawa, S. et al. (1987) Biochemical and Biophysical Research Communications 142:511-518). Upon attachment to the cell membrane via a GPI anchor, a 26-amino-acid C-terminal sequence, called the M-domain, is cleaved. This protein is heavily glycosylated, leading to a final weight of 180 kD. Amino acid sequence analyses have shown that CEA is a member of the immunoglobulin supergene family. CEA has an N-terminal N domain of 108 amino-acids that is similar to the immunoglobulin variable domain IgV, and then six domains, A1-B1-A2-B2-A3-B3, that are similar to the immunoglobulin constant domain IgC. Each A-B pair comprises 178 amino acids. Each domain of CEA is encoded by a separate exon. All known CEA family members that are translated have an N domain; they differ in the number and organization of IgC-like domains, the presence or absence and method of membrane attachment, and the presence or absence of a cytoplasmic domain. CEA does not have a cytoplasmic domain. Proteins of the CEA family are highly glycosylated.
The human CEA gene family currently consists of thirty genes (CEACAM1, CEACAM3-CEACAM8, AG128375, PSG1-PSG11, and CEACAM-ps1-CEACAM-ps11) and is divisible into three subgroups: thirteen CEA cross-reacting genes of which eight (CEACAM1, CEACAM3-CEACAM8 and AG128375) are expressed Frangsmyr, L. (1999) Tumor Biology 20:217-292, and Potera, C. (2001) Genetic Engineering News 21(12):9), eleven pregnancy-specific glycoproteins (PSG1-PSG11) of which at least nine expressed, and six unexpressed pseudogenes. PSG Accession Numbers include: M20882, M23575, M25384, U18467, U25988, M21822, M17908, M20879, J04539, M34715, M33665, U18469, M33666, M31125, X17610, U25987, and M31126. Only some CEA family members are present in primates, suggesting that the CEA family is undergoing strong selection and rapid evolution.
The CEA subfamily of cross-reacting antigens in humans, includes eight expressed genes including: CEACAM1 (CEACAM1-4L for biliary glycoprotein or BGP), CEACAM3 (CEACAM3-1L), CEACAM4 (CEACAM4), CEACAM5 (carcinoembryonic antigen or CEA), CEACAM6 (non-specific cross-reacting antigen or NCA), CEACAM7 (CEACAM7-2), CEACAM8 (CEACAM8) and AG128375. CEACAM1 (CEACAM1-4L) encodes thirteen splice variants (Genbank Accesion Numbers: D12502, X16354, X14831, S71326, X16356, D9031 1, D90312, D90313, E03350, E03351, E03352, M76742, M76743, D12502, and M76744) that contain zero to three IgC-like domains, membrane-spanning domains with cytoplasmic domains in some, with others not attached to the membrane, and two with additional extracellular Alu repeat sequences (CEACAM5 has Alu repeats that are untranslated). The CEACAM1 proteins range in size from about 80 kd to about 160 kd. CEACAM3 encodes three splice variants (Genbank Accession Numbers: L00692, D90277, D90278 and L00693) that contain no IgC-like domains; one variant is not attached to the membrane and two are attached with hydrophobic membrane-spanning domains and cytoplasmic domains. CEACAM7 encodes two splice variants (Genbank Accession Numbers X98311 and AF006623), one of which has one IgC-like domain (the other has none), with both attached via GPI anchors. CEACAM4-6 and -8 are not known to have splice variants. CEACAM4 (Genbank Accession Number D90276) encodes one IgC-like domain and is attached with a membrane spanning domain and a cytoplasmic domain. CEACAM5, CEA, is described above. CEACAM6 (NCA) encodes one AB pair of IgC-like domains and is attached via a GPI anchor. NCA (Genbank Accession Numbers M29541 and M18728) is present in two forms, 50 kD and 90 kD. CEACAM8 (CEACAM8) encodes an AB pair of IgC-like domains and is attached via a GPI anchor. The CEACAM8 protein is about 95 kD (Hammarstrom, S. et al. (1997) “The Nature and Expression of the Human CEA Family” in Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives, Stanners, C. Ed., pp 1-30, Harwood Acad. Publishers, Amsterdam).
CEA and CEACAM7 are expressed in epithelial cells, CEACAM3 and CEACAM8 in granulocytic cells, and CEACAM1 and CEACAM6 in many cell types, including epithelial cells. CEA is expressed in columnar epithelial cells and goblet cells of the colon, in mucous neck cells and pyloric mucous cells in the stomach, in squamous epithelial cells of the tongue, esophagus and cervix, in secretory epithelia and duct cells of sweat glands, and, in epithelial cells of the prostate (Hammarstrom, S. (1999) Seminars in Cancer Biology 9:67-81).
In situ hybridization, immunohistochemistry, and immunoelectron microscopy show CEACAM5 (CEA) and CEACAM6 (NCA) to have very similar expression patterns (Frangsmyr, L. (1999) Tumor Biology 20:277-292). An example of a tissue that expresses NCA and not CEA is squamous carcinoma of the lung. Mature NCA is 310 amino acids, after a 34 amino acid leader peptide is cleaved. BLAST analysis of CEA and NCA mature proteins demonstrates that NCA has about 86% amino acid identity with CEA (Thompson, J and Zimmermann, W. (1988) Tumor Biology 9:63-83).
In May 2001 a new cancer antigen, the eighth expressed member of the CEA cross-reacting subgroup, was cloned by AlphaGene. AG128375 was reported in the press as a splice variant of CEA Potera, C. (2001) Genetic Engineering News 21(12):9-10), but actually it is a completely new gene that is a member of the CEA gene family. It resides on a different chromosome from CEA and is prostate specific, Like CEA, AG128375 has an N domain and 6 IgC-like domains in the same order. AG128375 is expressed in normal and cancerous prostate tissue as well as in prostate tissue that has metastasized to the bone marrow, as shown by testing prostate-derived metastatic bone marrow cell lines. AG128375 is approximately the sane size as CEA and is likely to be similarly glycosylated, but it is expressed in a different distribution of tissues.
Although CEA has only been identified in humans, other members of the CEA gene family have been shown to exist in other species. CEA family members have been identified in mice, rats, guinea pigs, and non-human primates (http://www.med.uni-muenchen.de/cea/contents/introduction.htm).
Currently, colorectal cancer is the second most prevalent cancer in the United States and the presence or absence of CEA plays multiple roles in the fight against cancer. CEA is considered a tumor marker, but because it is expressed in so many normal and benign tissues, it is not a useful marker for cancer screening. However, CEA is a useful marker for predicting a patient's response to a therapy or tracking a patient's response throughout a therapy. In clinical medicine, CEA serum levels can assist in detecting advanced colorectal cancer as well as other various cancers. CEA levels generally increase with well-differentiated tumors and little increase is noted in poorly-differentiated adenocarcinomas. CEA blood concentration is used for monitoring cancer treatment. Patients determined to have high CEA levels before surgery always return to normal range post-operatively. An increase in the CEA level after successful surgery has been shown to indicate reoccurrence of the tumor (Wirakapun, S. (2001) Diseases of the Colon and Rectum 44:231-235; Miles, W. F. A (1995) Br. J. of General Practice 45:287-288; and Chu, D. Z. J. (1991) Archives of Surgery 126:314-316).
Assay methods for CEA are reported in the following publications. U.S. Pat. No. 4,180,556 (issued Dec. 25, 1979) reports a pretreatment method for CEA. Pretreatment of CEA, before immunoassays, comprises adding perchloric acid (PCA) to the sample, adding buffered source of potassium ions to precipitate the potassium perchlorate, and centrifuging to remove precipitate, thereby eliminating the need for dialysis after PCA treatment. U.S. Pat. No. 4,272,504 (issued Jun. 9, 1981) reports an antibody adsorbed support assay for CEA. U.S. Pat. No. 4,299,815 (issued Nov. 10, 1981) reports an improved process for radioimmunoassay of CEA. U.S. Pat. No. 4,349,528 (issued Sep. 14, 1982) reports an antibody specific for the 180 kd CEA. U.S. Pat. No. 4,818,709 (issued Apr. 4, 1989) reports an immunoassay for CEA and kits for such an assay. U.S. Pat. No. 4,863,853 (issued Sep. 5, 1989) reports a method of determining the value of monitoring CEA levels in patients undergoing therapy. U.S. Pat. No. 5,200,316 (issued Apr. 6, 1993) reports immunoassay methods using noncross-reactive CEA family antibodies. U.S. Pat. No. 6,013,772 (issued Jan. 11, 20000) reports CEA family antibodies and uses thereof. EPA 0343946 (published Nov. 29, 1989) reports synthetic CEA fragments that include a unique epitope, assays which utilize such fragments, and kits. Rogers, G. T. et al. (1981) Br. J. Cancer 44:371-380 describes a double antibody radioimmunoassay for CEA.
Members of the CEA gene family have been considered for use as vaccines, with hope that they might serve as antigenic targets for eliciting anti-cancer immune responses, for treatment and/or prevention. CEA family member proteins may play a role in cell aggregation, possibly as intercellular adhesion molecules, suggesting that vaccines targeting CEA family member proteins may be particularly useful for preventing metastasis. For CEA family member proteins to be used as vaccines, there must be sources of CEA family member proteins of sufficient purity and quantity. For this reason, as well as for basic research, there has been a long history of attempts to purify CEA family member proteins.
The first attempts to purify CEA family member proteins utilized a glycoprotein solvent extraction step followed by one or several forms of size-exclusion chromatography. The solvent of choice was initially PCA. Krupey, J. et al. (1967) Nature 215:67-68 describes the purification of CEA from the digestive system. CEA was purified by PCA extraction, paper block electrophoresis, and size-exclusion chromatography with Sephadex G-200. Coligan, J. E. (1972) Immunochemistry 9:377-386 describes isolation and characterization of CEA. CEA was isolated by PCA extraction, filtration, and size-exclusion chromatography using Sepharose 4B and Sephadex G-200. Krupey, J. (1972) Immunochemistry 9:617-622 describes purifying CEA from tumor tissue. CEA was purified by PCA extraction, sequential size-exclusion filters, gel-filtration chromatography, and size-exclusion chromatography using Sepharose 4B, Sephadex G-25, and Sephadex G-200. Banjo, C. et al. (1974) Cancer Research 34:2114-2121 describes the intermolecular heterogeneity of CEA. Purification of CEA was performed by the method of Krupey (1972). Laurence, D. J. R. et al., (1975) Br. J. Cancer 32:295-299 describes the first British Standard for CEA, prepared by PCA extraction and size-exclusion chromatography by Sepharose 4B and then Sephadex G-200. Matsuoka, Y. et al, (1975) Immunochemistry 12:779-782 describes attempts to identify anti-CEA antisera that was specific to the tumor-expressed form of CEA. CEA was partially purified by PCA extraction, ethanol precipitation, size filtration by successively smaller filters, and size-exclusion chromatography with Sepharose 4B. Kuroki, M (1981) Cancer Research 41:713-720 describes the purification and characterization of CEA from human feces. Purification of CEA was performed by the method of Krupey (1967) with additional gel filtration. Matsuoka, Y. (1976) “Cancer Specificity Problems of Carcinoembryonic Antigen” from Proceedings of the Euro. Econ. Comm. Symposium, pp 3-14 describes a CEA from human feces. CEA was prepared by PCA extraction, ethanol fractionation, and size-exclusion chromatography with Sepharose 4B. U.S. Pat. No. 4,871,834 (issued Oct. 3, 1989) reports monoclonal antibodies specific to CEA, the processes of making such antibodies, and the use of such antibodies. The antigenic CEA was prepared by PCA extraction followed by three steps of size-exclusion chromatography using Sepharose 4B, Sepharose 6B, and Sephadex G200. U.S. Pat. No. 4,228,236 (issued Oct. 14, 1980) reports CEA recombinant cell lines and recovering CEA from such cell lines utilizing PCA extraction.
There was controversy as to the effect of PCA, a strong acid, on the carbohydrate moieties on CEA family member proteins. Several groups attempted to use other methods to separate CEA family member proteins from cellular materials by shortening the PCA step or eliminating it altogether. Several different glycoprotein extraction or precipitation chemicals were utilized. Kimball, P. M. and Brattain, M. G. (1978) Cancer Research 38:619-623 describes a comparison of CEA purification methods. CEA was isolated by 1) saline-isolation, 2) saline-isolation and PCA extraction, or 3) saline-isolation and dialysis at pH 5 and heat treatment. Additional isolation procedures followed. Approximately 90% of the CEA antigenic activity was lost from each peak (identified with isoelectric focusing) after treatment with PCA. Carrico, R. J. et al. (1975) Cancer Research 35:2928-2934 describes a change in antigenicity of CEA with PCA extraction. CEA was isolated by Tris-acetate extraction at pH 6.9, anion-exchange chromatography, affinity chromatography with Con A, and size-exclusion chromatography with Sepharose 4B and Sephadex G200. Ritschard, W. J. (1983) Experientia 39:375-377 describes CEA purification without PCA. CEA was separated from other cellular components by polycarbonic acid extraction, followed by other techniques. Tu, Y. Y. et al. (1988) Tumor Biology 9:212-220 describes the purification of CEA from GW-39, a xenografted human colonic tumor system in hamsters. CEA was purified away from other cellular materials by acetic acid extraction followed by additional techniques. Duraiswami, S. et al. (1976) Intl Research Comm. Med. Science 4:172 extracted CEA with an 0.05M acetic acid-sodium acetate buffer at pH 4.5 also containing 40% ethanol, followed by acetone precipitation, ammonium sulfate extraction and precipitation, and size-exclusion chromatography at pH 4.0. Matsuoka, Y. et al, (1975) Immunochemistry 12:779-782 describes attempts to identify anti-CEA antisera that was specific to the tumor-expressed form of CEA. CEA was prepared by PCA extraction, ethanol precipitation, and other procedures. Rule, A. H. and Goleski-Reilly, C. (1973) Br. J. Cancer 28:464-468 and Rule, A. H. and Goleski-Reilly, C. (1974) Cancer Research 34:2083-2087 describe CEA fingerprints of various tissues. CEA was prepared by saline extraction, urea glycoprotein dissociation, and sucrose gradient electrofocusing chromatography. Radio-immunoassay (RIA) showed a change in CEA fingerprints with the use of PCA. Duraiswami, S. et al. (1976) “An evaluation of some methods for the isolation of CEA” in P. Franchimont Ed. Cancer Related Antigens pp 23-35, North Holland Publishing Co. and Duraiswami, S. et al. (1976) IRCS Med Sci: Immunol 4:172 describes an improved procedure for isolating CEA comprising: ethanol-acetate extraction, solubilizing the acetone powder with two extractions of distilled water, (NH4)2SO4 fractionation, and two cycles of size-exclusion chromatography with Sephadex G200 at pH 4.0. Extracting with PCA resulted in a 75% loss of CEA antigenic activity. Rosai J. et al. (1972) Int. J. Cancer 10:357-367 describes CEA purification by extraction with 0.25M sucrose and TE (0.05M:0.002M), precipitation with 0.1M LiCl, extraction with 0.3M lithium diiodosalicylate at pH 7.5, extraction with 25% phenol, and precipitation with ethyl alcohol. Meltzer, M. S. et al. (1971) J. Nat. Cancer Inst. 47:703-709 describes the isolation of tumor-specific antigens by extraction in 3M hypertonic KCl, extraction with 0.1M NaCl, precipitation with 2M ammonium sulfate, and size-exclusion chromatography with Sephadex G200.
Lectin affinity-chromatography was also utilized, to further prepared CEA family member proteins (Chu, T. M. et al (1974) Cancer Research 34:212-214) or to avoid harsh chemical such as PCA. This approach was limited because many of the proteins isolated with CEA are also glycoproteins. Rogers, G. T. (1974) Nature 251:519-521 describes CEA isolated by Con A chromatography. CEA was isolated by the methods of Krupey (1972) and then by affinity chromatography using Con A Sepharose. Boenisch, T. and Nørgaard-Pedersen, B. (1975) Clinica Chimica Acta 60:51-57 describes two CEA variants, one reactive with Con A and the other not reactive with Con A, separated by Con A affinity chromatography. CEA was extracted by PCA extraction, size-exclusion chromatography with Sephadex G-200, affinity chromatography with Con A Sepharose, followed by an optional repeat of size-exclusion chromatography with Sephadex G-200. Rogers, G. T. et al. (1976) Br. J. Cancer 33:357-362 describes isolation of CEA. CEA was purified by Coligan, J. E. (1972) Immunochemistry 9:377-386 and affinity chromatography with Con A Sepharose, twice. Slayter, H. S. and Coligan, J. E. (1976) Cancer Research 36:1696-1704 describes CEA fractionated with Con A chromatography. CEA was fractionated by PCA extraction, size-exclusion chromatography with Sepharose 4B and Sephadex G-200, and affinity chromatography with Con A Sepharose. Egan, M. L. et al. (1977) Cancer Research 37:2638-2643 describes isolation of CEA-like substances from healthy colon tissue. CEA-like material was isolation by PCA extraction, size-exclusion chromatography with Sepharose 6B and Sephadex G-200, and affinity chromatography with Con A. Pritchard, D. G. and Egan, M. L. (1978) Immunochemistry 15:385-387 describes an improved procedure for isolating CEA. CEA was isolated by PCA extraction, ethanol precipitation, size-exclusion chromatography with Sepharose 4B, and affinity chromatography with Con A-Sepharose. Matsuoka, Y. et al. (1978) Int. J. Cancer 21:604-610 describes proteolytic release of antigenic fragments from CEA corresponding to cross-reacting antigens. CEA was prepared by PCA extraction, decreasing size membrane filtrations, size-exclusion chromatography with Sepharose 4B, 6B and Sephadex G-200. Another batch was prepared further by affinity chromatography with Con A Sepharose 4B. Coligan, J. E. and Slayter, H. S. (1979) Molecular Immunology 16:129-135 describes characterization of Con A purified CEA. A new method for purifying CEA was attempted because other references suggested that PCA purification was too harsh and alters the physical, chemical, and immunological properties of CEA. The tumor tissue used to purify CEA was homogenated in a PBS solution, dialyzed and lyophilized. CEA was purified from this material by Sepharose 4B chromatography twice, Sephadex G-200 chromatography, and then Con A Sepharose chromatography. Hill, R. (1981) Ajebak 59:469-476 describes CEA characteristics when extracted from different sources. CEA was prepared by PCA extraction, size-exclusion chromatography with Sephadex G-200, affinity chromatography with Con A, affinity chromatography with wheat germ lectin Sepharose 6 MB, and isoelectric focusing. CEA peaks were identified at pI3.2 and pI 4.4. Watt, S. M. et al. (1991) Blood 78:63-74 describes a neutrophil-specific epitope expressed by members of the CEA family. CEA was prepared by PCA extraction, size-exclusion chromatography with Sepharose 6B, and affinity chromatography with Con A sepharose. Harvey, S. R. and Chu, T. M. (1975) Cancer Research 35: 3001-3007 describes two molecular variants of CEA. CEA was separated by PCA extraction, affinity chromatography with Con A Sepharose, and size exclusion chromatography with Sephadex G-200. Keep, P. A. et al. (1978) Br. J. Cancer 37:171-182 describes the extraction of CEA and the effect of PCA. CEA was extracted by several versions of a PCA extraction, size-exclusion chromatography with Sepharose 6B and Sephadex G-200, and affinity chromatography with Con A Sepharose.
Immnuno-affinity chromatography using anti-CEA antibodies was used to even further purify CEA family member proteins. This approach was similarly limited as lectin affinity chromatography, because many proteins co-purifying with CEA share immunologic epitopes. Eveleigh J. W. (1974) Cancer Research 34:2122-2124 describes an isolation method that separates CEA from several proteins that had previously been isolated with CEA. These proteins were isolated by saline extraction, immuno-affinity chromatography, and anion-exchange chromatography at a range of salt concentrations, twice. At least eight peaks were observed. Previously generated “CEA” antibodies were made against all of these proteins. Acidic extraction with HCl at pH 3.0 yielded more of the basic components. PCA extraction yielded more of the acidic components. It was noted that size-exclusion chromatography would be ineffective at separating these components, as they are of similar molecular weights. Ashman, L. and De Young, N. J. (1977) Immunochemistry 14: 329-336 describes immunoadsorbent purification of CEA. Standard CEA was purified by PCA extraction, followed by size-exclusion chromatography with Sepharose 4B and Sephadex G200. Experimental CEA was purified by phosphate extraction, cotton filtration, and either immunoadsorbent precipitation with rabbit or goat anti-CEA antiserum (pH 6.5), or affinity chromatography with Con A. EPA 0102008 (published Mar. 7, 1984) reports CEA isolation, comprising PCA extraction, size filtration, anti-CEA affinity chromatography, and liquid chromatography. Price, M. R. et al. (1986) Cancer Letters 33:83-89 describes the association of the Y Hapten with CEA. Antigens were isolated with detergent and immuno-affinity chromatography. EPA 0212880 (published Mar. 4, 1987) reports production of CEA antigen comprising cultivating a CEA-producing cell line in monolayer culture with further cultivation steps, harvesting the medium containing the secreted CEA, and purification of CEA until iodination grade product was obtained. The purification steps comprised PCA and ammonium sulfate extractions, and immuno-affinity chromatography. CEA as a composition of matter having certain physical properties and immunoreactivity was reported. Tu, Y. Y. et al. (1988) Tumor Biology 9:212-220 describes the purification of CEA from GW-39, a xenografted human colonic tumor system in hamsters. CEA was purified by acetic acid extraction, affinity chromatography with anti-CEA, and size-exclusion chromatography with Sepharose Cl-6B. The purified CEA pI was 4.4. U.S. Pat. No. 5,672,513 (issued Sep. 30, 1997) reports CEA lacking the carboxyl terminal 26 amino acids and uses thereof. rCEA was purified by immuno-affinity chromatography, PCA extraction, and size-exclusion chromatography using Sephadex G-200 and Sepharose 6B. You, Y. H. et al. (1998) AntiCancer Research 18:3193-3202 describes the expression, purification, and characterization of a CEA minigene. Proteins were purified by PCA extraction and immuno-affinity chromatography.
Both types of affinity chromatography have been combined. Zimmerman, R. and Hammarström, S. (1978) Urological Res. 6:215-219 describes the isolation of CEA from bladder carcinoma. CEA was isolated by PCA extraction, size-exclusion chromatography with Sepharose 4B, and affinity chromatography with Con A and sheep anti-CEA serum. Santen, R. J. et al. (1980) Cancer Research 40:1181-1188 describes the partial purification of CEA from breast neoplasms. CEA was partially purified by 1) glass wool filtration, polyvalent immuno-affinity chromatography, wheat germ agglutinin lectin (alternative to Con A), Sepharose 4B affinity chromatography, hydroxylapatite precipitation, and ACA 34 gel filtration chromatography; 2) phenylmethyl sulfonyl fluoride/aminocaproic acid/EDTA/sodium azide proteolysis prevention step, monospecific immuno-affinity chromatography, and ACA 44 (34) chromatography; or 3) procedure 1) with the addition of a first proteolysis prevention step and also without lyophilization or freezing. Ritschard, W. J. (1983) Experientia 39:375-377 describes CEA purification without PCA. CEA was purified by polycarbonic acid extraction (not perchloric acid), size-exclusion chromatography with Sephadex G-200, and optionally affinity chromatography and/or immunoadsorption.
Anion-exchange chromatography has been an important tool for purifying CEA family member proteins. Anion exchange has been most common. Mistretta, A. P. et al. (1974) Experientia 30:1209-1210 describes CEA isolation from colon cancer tissue and generation of antiserum. CEA was isolated by PCA extraction, anion-exchange chromatography with DEAE-cellulose at pH 6.75, and size-exclusion chromatography with Sephadex G-200. Plow, E. F. and Edgington, T. S. (1975) Int. J. Cancer 15:748-761 describes the characterization of a CEA isomer, CEA-S. CEA-S was isolated by PCA extraction, isoelectric focusing, anion-exchange chromatography using DEAE cellulose, size-exclusion chromatography with Sephadex G-200, and immuno-affinity chromatography with anti-CEA (to remove CEA). Isopyknic density gradient ultracentrifugation was used to characterize the protein. CEA was isolated by PCA extraction and size-exclusion chromatography with agarose. Carrico, R. J. and Usategui-Gomez, M (1975) Cancer Research 35:2928-2934 describes the isolation of CEA from tumor tissue at neutral pH. CEA was isolated by anion exchange chromatography with DEAE-cellulose at pH 6.9, affinity chromatography with Con A Sepharose, size exclusion chromatography with Sepharose 4B and Sephadex G-200. Alternatively CEA was isolated by PCA extraction. Sixty to seventy percent of the CEA in crude tumor extracts or neutral pH isolations was destroyed and/or becomes insoluble under acidic conditions. Fritsche, R. and Mach, J. (1977) Immunochemistry 14:119-127 describes CEA from normal colon mucosa. CEA was isolated by 1) PCA extraction, size-exclusion chromatography with Sephadex G-200 and Sepharose 6B, and anion-exchange chromatography with DEAE at pH 7.4; or 2) by steps 1-2 above with the Sepharose 6B and anion-exchange chromatography optional; or 3) by steps 1-2 above followed by immuno-affinity precipitation with goat anti-CEA and another PCA extraction; or 4) by steps 1-2 above followed by immuno-affinity chromatography with goat anti-CEA. Lisowska, Z. et al. (1979) Neoplasma 26: 157-167 describes the purification of CEA from three different sources. CEA was purified by PCA extraction, size exclusion chromatography with Sepharose 4B and Sephadex G-200, affinity chromatography with Con A, and anion-exchange chromatography with DEAE. U.S. Pat. No. 4,145,336 (issued Mar. 20, 1979) reports the isolation and utilization of a CEA isomer. CEA-S1 was isolated by PCA extraction, brought to pH 3-6, and iso-electric-focused. The peak at pI 4.4-4.6 was recovered, separated by size-exclusion chromatography (A-1.5 fine agarose and Sephadex G-200), further isolated by ion exchange chromatography (DEAE cellulose), and contaminants removed by immuuno-affinity chromatography. Isopyknic density gradient ultracentrifugation was also performed. U.S. Pat. No. 4,140,753 (issued Feb. 20, 1979) reports a radioimmunoassay for an isolated CEA isomer. CEA-S1 was isolated by PCA extraction, brought to pH 3-6, and iso-electric-focused. The peak at pI 4.4-4.6 was recovered, separated by size-exclusion chromatography (Sephadex G-200), further isolated by ion exchange chromatography (DEAE cellulose at pH 8), and contaminants removed by immuno-affinity chromatography. Isopyknic density gradient ultracentrifugation was also performed. Kuroki, M. et at. (1982) J of Immunological Methods 60:221-233 describes antigenic reactivities of several preparations of purified CEA and related antigens. Antigens were extracted by the methods of Matsuoka, Y. et al, (1975) Immunochemistry 12:779-782) and Matsuoka, Y. et al. (1978) Int. J. Cancer 21:604-610. A combination of procedures including PCA extraction, affinity chromatography, size-exclusion chromatography (gel filtration, Sepharose 6B, Sephadex G-200, Sephadex G-100), and anion-exchange chromatography were used. Hedin, A. et al (1986) Molecular Immunology 23:1053-1061 describes monoclonal antibodies to CEA. CEA was purified by PCA extraction, anion exchange chromatography with DEAE Sephadex, size-exclusion chromatography with Sepharose 4B, affinity chromatography with Con A Sepharose, and size-exclusion chromatography with Sephadex G-200 or CEA was purified with immuno-affinity chromatography. Ford, C. H. J. et al. (1987) Tumor Biology 8:241-250 describes purifying CEA with monoclonal antibody immunoadsorbent precipitation. Conventional CEA was purified by PCA extraction, clarification with glass fiber filters, ethanol precipitation, phosphate resuspension, size-exclusion chromatography with Sepharose 4B and Sephacryl S300, anion-exchange chromatography with DEAE Sephacel at pH 8.3, and affinity chromatography with Con A Sepharose. Experimental CEA was purified by PBS extraction, buffer exchange with Sephadex G25, and immuno-affinity chromatography with 11-285-14 monoclonal anti-CEA antibody. Krupey, T. et al. (1974) Specialia 30:1209-1210 describe a method for purifying CEA comprising: PCA extraction, extraction in 3M KCl, 0.05M NaH2PO4 at pH 4.2, anion-exchange chromatography at pH 6.75, and size-exclusion chromatography at pH 4.5.
WO 95/32286 (published Nov. 30, 1995) reports recombinant CEA, lacking the transmembrane domain, expressed in insect cells using a baculovirus expression system, and methods of purifying such secreted CEA. Purification methods comprise chromatography utilizing four resins: hydrophobic interaction HIC ether 650 m, HIC Butyl 650M, lentil lectin Sepharose 4B, and DEAE (anion chromatography) Sepharose Fast Flow. Before chromatography, insect cell supernatant was acidified with acetic acid to pH 3.5 and incubated for one hour, in order to inactivate all viruses. The supernatant was then neutralized to pH 7.0 and filtered to remove precipitated viral and cellular proteins. rCEA was said to remain soluble throughout this step. The remainder of the purification process was carried out at neutral pH. The application also demonstrates the use of purified CEA (from Vitro Diagnostics) as a standard, for comparison of physical properties and for comparison of utility as a vaccine.
Mixed-bed ion-exchange chromatography using both anion- and cation-exchange resins has been used occasionally for the purification of CEA family member proteins. U.S. Pat. No. 3,867,363 (issued Feb. 18, 1975), U.S. Pat. No. 3,956,258 (issued May 1, 1976), U.S. Pat. No. 4,086,217 (issued Apr. 25, 1978), and U.S. Pat. No. 4,180,499 (issued Dec. 25, 1979) report methods for isolating, identifying, and detecting CEA material. CEA material was isolated by glycoprotein solvent extraction, gel-filtration, and mixed bed ion-exchange chromatography by eluting in ammonium acetate at pH 4. PCA extraction was preferred. Component A and component B, the isolated CEA material, range from 200-500 kD. Newman, E. S. et al. (1974) Cancer Research 34:2125-2130 describes isolation of CEA and CCA-III by PCA extraction, mixed-bed ion-exchange chromatography at pH 4.0, and size-exclusion chromatography with Sepharose 6B. Two forms of CEA, 180 kD and 200 kD were isolated together. CCA-III was reported to have contaminated the CEA preparation, by no more than 0.5%. CEA also was reported to have contaminated the CCA-III preparation, by less than 0.1%. WO 8402983 (published Aug. 2, 1984) reports CEA family antigens and antibodies and their methods of use. Antigens were purified by ethanol extraction, anion-exchange chromatography with DEAE cellulose, size-exclusion chromatography with Sephacryl S-300, and affinity chromatography with a specific anti-CEA antibody and then with a cross-reacting anti-CEA antibody. CEA, NCA, and Meconium Antigen (MA) were also purified by PCA extraction, mixed-bed ion-exchange chromatography equilibrated to 0.1 M ammonium acetate (NH4Ac) pH 4, size-exclusion chromatography with Sepharose 6B and Sephadex G-200, and affinity chromatography with Con A Sepharose. Radiolabeled CEA was used as a marker.
Grunert, F. et al. (1984) Tumor Biology 5:221-232 teaches the use of cation-exchange chromatography to isolate CEA, but the method failed to purify CEA away from two “sticky” co-purifying proteins. The pH at which this procedure was carried out was not stated. The proteins were purified by PCA extraction, size-exclusion chromatography with Sepharose 4B and Sephadex G-150, affinity chromatography with Con A, and further purification steps chosen from: Octyl-Sepharose, DEAE-Sepharose, CM-Sepharose, Heparin-agarose, or Hydroxyapatite chromatography. The “sticky” proteins were about 45 and 58 kD.
Sheehan, D. and FitzGerald, R. (1996) Chap 14: Ion-Exchange Chromatography in “Methods in Molecular Biology: Protein Purification Protocols,” Doonan, S. ed. Humana Press, New Jersey, teaches that the best approach to developing a new purification method using ion-exchange chromatography was to determine the binding of the protein of interest on both cation- and anion-exchange resins at a range of pH values. When mixed-bed ion exchange chromatography or cation-exchange chromatography was used to purify CEA family member proteins, the resulting purified proteins were not substantially free of cross-reacting antigens. When CEA family member proteins were placed in acidic solutions, sometimes they precipitated. Also, when chromatography utilizing cation-exchange resin was used, glycosylation of the protein of interest was first modified by PCA or other chemicals.
Cation-exchange chromatography was used to isolate nagase-digested CEA peptides (Banjo, C. (1974) Int. J. Cancer 13:151-163). CEA was purified by the method of Krupey, J. (1972) Immunochemistry 9:617-622 and digested with neuraminidase which decreased the carbohydrate content by over 10%, removed all of the sialic acid, and significantly reduced fucose and galactose, before digestion with nagase and cation-exchange chromatography. Chromatography was performed at pH 3.1.
Other methods known to the art have been used to purify CEA family member proteins, such as iso-electric focusing and HPLC. Hill, R. et al. (1981) Molecular Immunology 18:647-653 describes CEA purified from malignant ascitic fluid of an ovarian cancer. CEA was purified by heat treatment, PCA extraction, size-exclusion chromatography with Sephadex G150, affinity chromatography with Con A, and isoelectric focusing. Hefta, L. et al. (1992) Cancer Research 52:5647-5655 describes expression of CEA and predicted immunogenic epitopes in HeLa cells. Expressed products were purified by affinity chromatography using monoclonal antibodies specific for each domain and by reverse phase HPLC. CEA has also been purified by phosphatidlyinositol-specific phospholipase C (PI-PLC) extraction, utilizing knowledge that CEA was bound to the cell membrane by a GPI moiety (Matsuoka, Y. et al. (1991) Tumor Biology 12:91-98). Tissues were minced, homogenized, and digested with PI-PLC; size-exclusion chromatography with Sepharose 6B at pH 5.2 was performed on the supernatant. Some samples were treated with PCA before chromatography.
Many publications of new methods for purifying CEA are closely followed by further publications describing impurities of the purified products. See Rogers, G. T (1976) Biochimica et Biophysica Acta 458:355-373, Coligan, J. E. et al. (1973) Immunochemistry 10:591-599, Aitio, M. et al. (1978) FEBS Letters 93:29-32, Pusztaszeri, G. and Mach, J. (1973) Immunochemistry 10:197-204, Grunert, F. et al. (1984) Tumor Biology 5:221-232 (including PCA extraction, size-exclusion chromatography, Con A affinity chromatography, anion- and cation-exchange chromatography, heparin-agarose, and hydroxyapatite chromatography), WO 8402983 (published Aug. 2, 1984) and Ford, C. H. J. et al. (1987) Tumor Biology 8:241-250 (immuno-affinity chromatography).
Methods for purifying CEA family members have been inadequate at obtaining the protein of interest. Some of the methods, such as extraction with PCA, modified the protein of interest during the purification process. Methods for purifying individual CEA family members away from other CEA family members and other contaminants have been inadequate because so many of the proteins are physically similar. The chemical extraction and precipitation steps isolated many chemically similar proteins together. Lectin affinity steps isolated many glycosylated proteins together. Immuno-affinity steps isolated many cross-reacting antigens together. Ion-exchange chromatography isolated many similarly charged proteins together. Size-exclusion chromatography isolated many similarly sized proteins together. PI-PLC digestion isolated many GPI-membrane-bound proteins together.
None of the previously-available methods for purifying CEA family member proteins has stood out as being superior to the others. Recent applications no longer even mention a purification scheme as preferable. WO 0155337 (published Aug. 2, 2001) reports purifying a CEA-like peptide by any method known to the art.
One need in the art for pure CEA family member proteins has been for use as a vaccine. The possibility of using CEA as a vaccine has been addressed in the literature for over a decade. Attempts to use purified CEA as a vaccine have been hampered by the difficulty of obtaining sufficient quantities of sufficiently pure CEA. No method has been available for sufficiently purifying CEA, without the use of PCA, away from other cross-reacting antigens.
It has proven difficult to obtain sufficient CEA from many recombinant culture systems. High amounts of a recombinant CEA have been obtained with a baculovirus expression system. WO 95/32286 (published Nov. 30, 1995) teaches expressing CEA in insect cells using a baculovirus vector. The baculovirus CEA (bvCEA) was expressed at very high levels, secreted, and easy to purify because of its abundance and lack of physically, chemically, and immunologically related proteins. The bvCEA was used successfully as a vaccine to treat mice. WO 95/32286 (published Nov. 30, 1995) reports recombinant CEA, lacking the transmembrane domain, expressed in insect cells using a baculovirus expression system, and methods of purifying such secreted CEA. Purification methods comprise chromatography utilizing four resins: hydrophobic interaction HIC ether 650 m, HIC Butyl 650M, lentil lectin sepharose 4B, and DEAE (anion chromatography) Sepharose Fast Flow. Before chromatography, insect cell supernatant was acidified with acetic acid to pH 3.5 and incubated for one hour, in order to inactivate any virus. The supernatant was then neutralized to pH 7.0 and filtered to remove precipitated viral and cellular proteins. rCEA was said to remain soluble throughout this step. The remainder of the purification process was carried out at neutral pH. The publication also demonstrates the use of purified CEA (from Vitro Diagnostics) as a standard, for comparison of baculovirus CEA physical properties and for comparison, as a reference, of the utility of the baculovirus CEA as a vaccine.
CEA has also been cloned into a vaccinia virus vector. The vvCEA DNA vector was used directly as a vaccine. When administered to mice, it induced the generation of anti-CEA-specific T cells. Administration of vvCEA also reduced the growth of syngenic, CEA transformed, murine colon adenocarcinomas. Kaufman, H. et al. (1991) Int. J. Cancer 48:900-907 describes a recombinant Vaccinia Virus expressing CEA, and its use as a vaccine, and Kantor, J (1992) J. Nati. Cancer Inst. 84:1084-1091 describes a vvCEA vaccine. CEA was not purified. U.S. Pat. No. 5,698,530 (issued Dec. 16, 1997) reports recombinant CEA in a vaccinia virus vector, which expresses CEA on the surface of infected cells and can elicit an immune response against CEA. Methods of treating patients were included. No CEA was purified; crude lysates were used. Kass, E. et al. (1999) Cancer Research 59:676-683 describes the use of vvCEA as a vaccine in CEA-transgenic mice. The control CEA vaccine was from Vitro Diagnostics. A vvCEA vaccine has been shown to elicit an immune response in patients (Conry, R. M. et al. (1995) J. Immunother. 18:137). WO 124832 (published Apr. 12, 2001) reports CEA DNA in adenovirus and vaccinia virus vectors as pharmaceutical compositions used as vaccines to treat or prevent tumors.
These techniques have the disadvantage of including viral materials, along with CEA, in the vaccine. A vaccine uncontaminated with viral materials was preferable. Unfortunately, recent studies show that vaccines of purified CEA protein are not particularly effective. Likely this was due to the presence of CEA on normal cells, the body recognizing CEA as self, and the consequent dampening of a potential immune response. For example, when mice that express (human) CEA as a self-antigen were vaccinated with CEA protein, the CEA protein failed to elicit an immune response. However, vaccination of these mice with vvCEA elicited a strong immune response (Shlom, J. (2000) Carcinoembryonic Antigen (CEA) Peptides and Vaccines for Carcinoma in “Peptide-Based Cancer Vaccines,” Kast, M. ed., Landes BioSciences). When testing vaccines of recombinant CEA family members, the corresponding native CEA family member protein was used as a reference standard (WO 95/32286 (published Nov. 30, 1995) and Kass, E. et al. (1999) Cancer Research 59:676-683).
The CEA vaccines that include viral materials, such as lysates or vectors, have been useful for demonstrating the ability of the body to mount an immune response to CEA family member proteins. Because vaccines of pure, virally uncontaminated CEA are preferable, attempts to create CEA family member protein vaccines without viral contaminants have been directed towards improving the immunogenicity of these proteins.
The immune system's ability to respond to CEA has been further demonstrated by the isolation of CEA-specific T-cells. Attention has focused on the isolation of antigens recognized by T cells because T cells are significant mediators of tumor rejection. T cells recognize antigens as epitopes that are presented on the cell surface by HLA proteins, in humans. In attempts to identify possible T cell antigen epitopes within CEA, the CEA protein sequence was searched for peptides matching the consensus motifs for HLA-A2, the most common HLA allele. Six peptide sequences, CAP1-CAP6, ranging from nine to eleven amino acids in length, were identified that matched the consensus motif for HLA-A2 and had minimal homology to the analogous sequences in the cross-reacting antigens NCA and CEACAM1-4L. A seventh peptide, CAP7, was recognized as having the motif for binding to HLA-A3. Peptides CAP1 through CAP6 were shown to bind T2 cells, with CAP1 having the strongest binding. A 177-amino-acid CEA fragment containing the CAP1 epitopes was described in EP 0343946 (published Nov. 29, 1989).
CEA T cell epitopes have been identified for a variety of HLA alleles. Thirty-four CEA specific peptides have been identified that fit with a specified HLA-A*0301-binding motif, with a set of six peptides having high binding affinity for this allele. Approximately seventy-three peptides have been identified that match the HLA-A*0201 motif. Eleven peptides have been identified that bind to HLA-A24, an allele which occurs in 60% of the Japanese population. CEA epitopes have also been identified using anti-idiotype schemes. Two such peptides are LCD-2 and CEA-B.
CEA peptides have been demonstrated to elicit CTL in vitro. Such peptides include CAP1 and CAP2 against HLA-A2, as well as peptides recognized by HLA-A3 and HLA-A24. CEA peptides have not been very effective as vaccines (Arlen, P. et al. (2000) Cancer Immunology Immunotherapy 49:517-529).
Since it was known in the art that peptide engineering can be used to alter the immunogenicity of epitopes (Kersh, G. J. and Allen, P. M. (1996) J. Exp. Med. 184:1259-1268 and Cho, B. K. (1998) J. of Immunological Methods 220:179-188), attempts to create more potent vaccines targeted to CEA have included modifying CEA epitopes. A successfully modified epitope stimulates CTL more efficiently while retaining specificity for the native antigen. Recently work has been directed toward optimizing CEA immunogenic epitopes. Because HLA-A2 was the most common HLA allele and CAP1 demonstrated the strongest binding, CAP1 was investigated (Zaremba, S. et al. (1997) Cancer Research 57:4570-4577 and WO 99/19478 (published Apr. 22, 1999)) first. These publications describe and report the CAP1peptide at amino acid positions 571-579 (YLSGANLNL, SEQ ID NO:1) and amino acid variants at positions 6 and 7, and pharmaceutical compositions containing such peptides. These modified peptides, used as a vaccine, in comparison to the native peptide, increase the immune response to CEA as demonstrated in vitro. CAP1-6D is called an agonist, because it facilitates the propagation of T-cells. Antagonists decrease the immune response to the target. Antagonist peptides are said to possibly be useful in modulating autoimmune reactions. The modified peptide CAP1-6D has the same sequence as the CAP1peptide except that it is substituted with D at the 6th amino acid of the peptide (YLSGADLNL, SEQ ID NO:2). CAP1-6D is currently being tested as a vaccine by the N.I.H.
A control, or reference standard, in the CAP1-6D clinical trial is a native, pure CEA composition, of this invention. There is a need in the art for pure CEA, uncontaminated with cross-reacting antigens or endotoxins, not purified with PCA or antibody affinity, for use as a control or reference standard in vaccine trials (Kass, E. et al. (1999) Cancer Research 59:676-683 and WO 95/32286 (published Nov. 30, 1995). There is also a need in the art for purified CEA preparations used as calibration standards or reference antigens. For example, some assays that quantitate CEA in unknown samples utilize purified CEA as a reference. Purified CEA was utilized as a standard in the development of those assays. Purified CEA has also been used as a reference standard for analyzing physical properties (WO 95/32286, published Nov. 30, 1995) and antigenic properties (Matsuoka, Y. et al. (1991) Tumor Biology 12:91-98) of unknown samples. Labeled CEA is also useful for tracing unlabelled CEA through experiments (WO 8402983 (published Aug. 2, 1984)).
CEA-targeted vaccines are more effective with costimulatory molecule adjuvants such as the cytokine IL-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Shlom, J. (2000) Carcinoembryonic Antigen (CEA) Peptides and Vaccines for Carcinoma in “Peptide-Based Cancer Vaccines,” Kast, M. ed., Landes BioSciences, Georgetown, Tex.). Advantages have also been noted when using diversified prime and boost strategies, for example utilizing a CEA protein vaccine and separately a CEA viral vector vaccine. Using several different CEA-targeted vaccines, individually, on the same patient, has been shown to be very effective.
Carbohydrate antigen 19-9 (CA19-9), a gastrointestinal-cancer-associated antigen, is defined by a monoclonal antibody, which was made by immunizing with a human colon cancer cell line. CA19-9 is elevated in the sera of patients with malignancies of the gastrointestinal tract, especially those who suffer from pancreatic cancer. CA19-9 is also present in normal epithelial lining of the biliary tract, gastric mucosa, pancreatic duct and bronchial glands (Obayashi, Y. et al. (2000) Respiration 67:146-152). CA19-9 has been a contaminant in CEA purifications. CA19-9 is the eptitope recognized by antibodies generated against the Sialyl Lewis A protein (Obayashi, Y. (2000) Respiration 67:146-152), such as 1116-NS-19-9 from Fujirebio Diagnostics (Japan) or NCL-CA19-9 from Novocastra Laboratories Ltd (Newcastle upon Tyne, UK).
Many proprietary purifications of CEA family member proteins are and have been available on the market, but none are as pure as the CEA family member proteins of this invention. Purity, including absence of cross-reacting antigens, CA19-9, endotoxins and antibodies, is particularly important when CEA family member proteins are used for vaccines or vaccine reference standards in trials. It is important that purification schemes not utilize antibody affinity steps, because the antibodies then appear as contaminants in the final product. It is also important that purification schemes not use perchloric acid (PCA), because PCA has been show to physically modify the glycosylation of CEA family member proteins, and such methods yield physically and immunogenically altered proteins. ERFA Biotech of Montreal, Canada, sells a CEA product that is stated to be 100% pure, purified by water homogenization, perchloric acid extraction, ion-exchange, antibody affinity and gel filtration chromatographies. This method includes use of both perchloric acid and an antibody affinity step. TriChem Resources of West Chester, Pa., also sells a CEA product, 10690B, that is stated to be at least 95% pure, purified by a method including an antibody affinity step. Lee Scientific of St. Louis, Mo., also sells CEA products, but they are purified and purchased elsewhere.
CEA has a pI of about 4.5 (U.S. Pat. No. 4,145,336 (issued Mar. 20, 1979), U.S. Pat. No. 4,140,753 (issued Feb. 20, 1979), Hill, R. (1981) Ajebak 59:469-476, and Tu, Y. Y. et al. (1988) Tumor Biology 9:212-220), has been shown to precipitate in some acidic pH ranges, and has been shown to be partially degraded by highly acidic PCA. When cation-exchange chromatography was used to purify CEA in previously known methods, two “sticky” proteins would not purify away (Grunert, F. et al. (1984) Tumor Biology 5:221-232). Mixed-bed ion-exchange, using both cation- and anion-exchange resins, was successful at isolating CEA along with a large collection of proteins and was not able to separate them from each other (U.S. Pat. No. 3,867,363 (issued Feb. 18, 1975), U.S. Pat. No. 3,956,258 (issued May 11, 1976), U.S. Pat. No. 4,086,217 (issued Apr. 25, 1978)). Newman, E. S. et al. (1974) Cancer Research 34:2125-2130 describes preparing CEA and CCA-III (NCA) using PCA extraction, mixed-bed ion-exchange chromatography at pH 4, and size-exclusion chromatography, but two forms of CEA were isolated, and the CCA-III contaminated the CEA preparation and the CEA contaminated the CCA-III preparation. WO 8402983 (published Aug. 2, 1984) describes purifying NCA and CEA using mixed-bed ion-exchange chromatography at pH 3.1, however the glycosylation-modifying chemical PCA was used, and no purity was stated except that a single band appears on gels.